Title: The mechanism of RNA 5' capping with NAD+, NADH, and CoA
Authors: Jeremy G. Bird1,2, †, Yu Zhang2, †, Yuan Tian3, Landon Greene4, Min Liu5, Brian
Buckley5, Jeehiun K. Lee3, Craig D. Kaplan6, Richard H. Ebright2,*, Bryce E. Nickels1,*
Affiliations: 1Department of Genetics and Waksman Institute, Rutgers University, Piscataway,
NJ 08854, USA.
2
Department of Chemistry and Chemical Biology and Waksman Institute, Rutgers University,
Piscataway, NJ 08854, USA.
3
Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854,
USA.
4
Analytical and Bioanalytical Development, Bristol-Myers Squibb Company, One Squibb Drive,
New Brunswick, NJ 08903, USA.
5
Environmental and Occupational Health Sciences Institute, Rutgers University, Piscataway, NJ
08854, USA.
6
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX
77843, USA.
†
Equal contribution
*Corresponding authors; ebright@waksman.rutgers.edu, bnickels@waksman.rutgers.edu
1
Abstract: Certain RNAs carry 5' caps containing nicotinamide adenine dinucleotide
(NAD+/NADH) or coenzyme A (CoA). It has been proposed that NAD+/NADH and CoA caps
are added to RNAs post-transcriptionally, in a manner analogous to addition of 7methylguanylate caps. Here we show that NAD+/NADH and 3'-dephospho-CoA (dpCoA) are
incorporated into RNA 5' ends during transcription initiation, functioning as non-canonical
initiating nucleotides (NCINs). We further show promoter sequence determines the efficiency of
NCIN capping and that both bacterial RNA polymerase and eukaryotic RNA polymerase II can
incorporate NCINs during initiation. We report crystal structures of bacterial transcription
initiation complexes containing NAD+- and dpCoA-capped products. The results define the
mechanism and structural basis of NAD+/NADH- and CoA-capping and suggest "ab initio
capping" with NCINs occurs in all organisms.
2
Main text:
The chemical nature of the 5' end of RNA is a key determinant of RNA stability,
processing, localization, and translation efficiency (1, 2). Recently it has been shown that a
significant fraction of bacterial RNAs carry 5'-end structures reminiscent of the 5'
7-methylguanylate “cap” in eukaryotic RNAs. In particular, bacterial RNAs containing 5'-end
nicotinamide adenine dinucleotide (NAD+) and 5'-end coenzyme A (CoA) have been identified in
both Gram-negative and Gram-positive taxa (3-5).
The mechanism by which NAD+/NADH- and CoA-capping occurs has not been defined.
NAD+, NADH, and a derivative of CoA lacking the 3'-phosphate, 3'-dephospho-CoA (dpCoA),
each is a nucleotide that shares an adenosine-diphosphate substructure with adenosine
triphosphate (ATP) (Fig. 1A, red). Therefore, in principle, NAD+, NADH, and dpCoA could, like
ATP, serve as initiating nucleotides in de novo transcription initiation by a cellular RNA
polymerase (RNAP), and thereby be incorporated ab initio into RNA. According to this
hypothesis, NAD+, NADH, and dpCoA function as non-canonical initiating nucleotides (NCINs).
The NCIN hypothesis was considered and rejected in prior work (3, 4). In particular,
prior work attempted, but failed, to detect the incorporation of NAD+, NADH, and dpCoA into
RNA during transcription initiation. Accordingly, it has been proposed, and currently is believed,
that NAD+/NADH- and CoA-capping occurs post-transcriptionally through enzymatic processes
analogous to those responsible for 7-methylguanylate-capping (5, 6).
Here we re-open the NCIN hypothesis. We note that a technical issue in the assays in
prior work--i.e., the use of ATP concentrations likely to out-compete, and render undetectable,
transcription initiation by NAD+, NADH, and dpCoA--renders the negative results in prior work
non-determinative. We re-examine the NCIN hypothesis, using assays not affected by this
technical issue, and we show that NAD+, NADH, and dpCoA function as NCINs with cellular
RNAPs.
3
To assess whether NAD+, NADH, and dpCoA serve as NCINs for transcription initiation
we performed in vitro transcription experiments with E. coli RNAP and DNA templates
containing promoters for RNAs identified as NAD+-capped RNAs [PgadY and PrnaI (5); Figs. 1-2].
We performed reactions using ATP, NAD+, NADH, or dpCoA as initiating nucleotide and [α32P]CTP as extending nucleotide. In each case, we observed efficient formation of an initial RNA
product (Fig. 1B). RppH, which processes 5'-triphosphate and -diphosphate RNAs to 5′monophosphate RNAs (7), processed the product obtained with ATP, but had no effect on the
products obtained with NAD+, NADH, and dpCoA (Fig. 1B). Conversely, NudC, which
processes 5'-capped RNAs to 5'-monophosphate RNAs (5), had no effect on the product formed
with ATP, but processed the products obtinaed with NAD+, NADH, and dpCoA (Fig. 1B).
Liquid-chromatography/tandem-mass-spectrometry (LC/MS/MS) analysis confirms the detection
and structural assignment of the product of NAD+-mediated transcription initiation (Figs. 1C,S1).
We conclude that NAD+, NADH, and dpCoA function as NCINs for transcription initiation.
To assess whether initial RNA products formed with NCINs can be extended to fulllength RNA products, we performed in vitro transcription reactions using PrnaI linked to a 100-nt
transcribed region lacking A (Fig. 2A). We observed production of full-length products with
NAD+, NADH and dpCoA, and observed that the full-length products contained NudC-sensitive,
capped 5'-ends (Fig. 2B). Equivalent results were obtained using [32P]-NAD+ to visualize
products (Fig. 2C). We conclude NAD+, NADH, and dpCoA not only function as NCINs for
transcription initiation but also yield initial products that can be extended to full-length RNA
products, and we propose that NCIN-mediated transcription initiation is responsible for
production of NCIN-capped RNAs in vivo.
The proposal that NCIN-mediated transcription initiation mediates production of NAD+-,
NADH-, and dpCoA-capped RNAs in vivo predicts that the identity of the base pair at the
transcription start site affects the production of NCIN-capped RNAs. Because NAD+, NADH, and
dpCoA each has the same Watson-Crick base pairing preference as ATP (Fig. 1A), it is predicted
4
that NAD+-, NADH-, and dpCoA-capping occurs only for RNAs transcribed from promoters that
contain A:T at the transcription start site, +1 (+1A promoters). Consistent with this prediction, we
find that NAD+-, NADH-, and dpCoA-capped products are formed only with +1A promoters, and
not with corresponding +1G promoter derivatives, in vitro (Fig. 3B). Further consistent with this
prediction, we note that, although only 43% of promoters in E. coli are +1A promoters (8), 100%
(6/6) of promoters for full-length RNAs identified as enriched for NAD+-capping in vivo in E.
coli are +1A promoters (5; Fig. S2). We infer that NCIN-capping occurs only for RNAs
transcribed from +1A promoters, both in vitro and in vivo.
The proposal that NCIN-mediated transcription initiation mediates production of NAD+-,
NADH-, and dpCoA-capped RNAs predicts that other aspects of promoter sequence, in addition
to +1A at the transcription start site, also may affect the production of NCIN-capped RNAs.
Consistent with this prediction, we find that the relative efficiencies of NAD+-mediated initiation
and ATP-mediated initiation, (kcat/Km,NAD+)/(kcat/Km,ATP), differ over a 1-2 order-ofmagnitude range at different +1A promoters in vitro, with PrnaI and PgadY showing high relative
efficiencies and PN25 and PT7A1 promoters showing low relative efficiencies (Fig. 3C). Further
consistent with this prediction, levels of NAD+-capping differ over a >2 order-of-magnitude
range for RNAs transcribed from +1A promoters in vivo in E. coli, with RNAs transcribed from
PrnaI and PgadY showing the highest levels of NAD-capping among full-length plasmid-encoded
RNAs and full-length chromosome-encoded RNAs (Fig. S2; 5). We conclude that promoter
sequence determinants in addition to +1A at the transcription start site affect the efficiency of
NCIN-capping in vitro and in vivo.
The correlations between in vitro results and in vivo results for effects of +1A at the
transcription start site and for effects of other aspects of promoter sequence (Figs. 3,S2; 5)
support the proposal that NCIN-mediated transcription initiation is responsible for production of
NCIN-capped RNAs not only in vitro but also in vivo.
5
All cellular RNAPs are members of a protein family having conserved structures and
mechanisms (9-11). To assess whether eukaryotic RNAP II, like bacterial RNAP, can use NCINs
as intiating nucleotides, we performed in vitro transcription experiments with Saccharomyces
cerevisiae RNAP II using ATP, NAD+, or NADH as initiating nucleotide and [α32P]-UTP as
extending nucleotide (Fig. S3). The results matched those obtained with bacterial RNAP.
Initiation occurred with NAD+ and NADH, and the products were insensitive to RppH and
sensitive to NudC. We conclude that eukaryotic RNAP II can initiate transcription with NAD+
and NADH, and we suggest that ab initio capping with NAD+ and NADH may occur in
eukaryotes as in bacteria.
To define the structural basis of transcription initiation with ATP, NAD+, and dpCoA as
initiating nucleotides we determined crystal structures of the corresponding product complexes
(Fig. 4; Table S1). We soaked pre-formed crystals of catalytically competent Thermus
thermophilus RNAP-promoter open complex, RPo (12) with ATP and CTP, NAD+ and CTP, and
dpCoA and CTP; we collected X-ray diffraction data; and we determined structures (resolutions
of 3.3 Å, 3.0 Å, and 3.1 Å, respectively). The results indicate that, in each case, the initiating
nucleotide--ATP, NAD+, or dpCoA--and the extending nucleotide--CTP--were able to react in
crystallo to form the initial RNA product--pppApC, NAD+pC, or dpCoApC, respectively--and
that RNAP was able to translocate in crystallo by 1 bp relative to the nucleic acid scaffold,
yielding a complex in a post-translocated state poised for RNA extension (Fig. 4). The electron
density maps showed unambiguous connected density in the RNAP "i-1 site" and "i site" and
showed a vacant "i+1 site," demonstrating RNA product formation and RNAP translocation to
yield a post-translocated state (Fig. 4, left). The results show graphically that NAD+ and dpCoA
serve as initiating nucleotides in transcription initiation and yield RNA products that are
competent for RNAP translocation and poised for extension to longer RNA products. In addition,
the results define the interactions that the RNA products make with DNA and RNAP.
6
A crystal structure of a substrate complex for transcription initiation, consisting of RPo,
ATP as initiating nucleotide, and CMPcPP as extending nucleotide, previously has been reported
(13, 14). However, no crystal structure of an initial product complex for transcription initiation
previously has been reported. The crystal structure of the initial product complex obtained herein
for intiation with ATP and CTP (RPo-pppApC) shows ordered density for all non-hydrogen
atoms of pppApC (Fig. 4A, left). The pppA residue of pppApC base pairs to the DNA template
strand in the RNAP active-center "i-1 site" and interacts, through its α-phosphate, with the same
RNAP residues that interact with the γ-phosphate of the initiating nucleotide in the substrate
complex (βQ688 and βH1237; residues numbered as in E. coli RNAP; 13, 14) and, through its βand γ-phosphates, with βR529 and βK1242 (Fig. 4A, right). The pC residue of pppApC base pairs
with the DNA template strand in the RNAP active-center "i site," making interactions, through its
sugar and phosphate, with the RNAP catalytic Mg2+ ion [Mg2+ (I)], and RNAP residues, notably
βK1065 and βK1073, that are identical to those made by the sugar and α-phosphate of the
initiating nucleotide in the substrate complex (Fig. 4A, right; 13, 14). Strikingly, the organization
of the RNAP active center enables the same RNAP residues (βQ688 and βH1237) to interact with
α-phosphate of the initiating nucleotide in the product complex and the γ-phosphate of the
initiating nucleotide in the substrate complex and enables the same RNAP residues (βK1065 and
βK1073) to interact with the α-phosphate of the extending nucleotide in the product complex and
the α-phosphate of the initiating nucleotide in the substrate complex.
The crystal structure of the initial product complex obtained upon initiation with NAD+
and CTP (RPo-NAD+pC) shows ordered density for all non-hydrogen atoms of NAD+pC (Fig.
4B, left). The NAD+ residue of NAD+pC base pairs to the DNA template strand in the RNAP "i-1
site," making interactions through its base and phosphates identical to those in the product
complex for initiation with ATP (Fig. 4 A-B, right), and making additional interactions through
its nicotinamide ribonucleoside moiety with RNAP βD516 and βH1237 (Fig. 4B, right). The pC
7
residue of NAD+pC base pairs to the DNA template strand in the RNAP active-center "i site,"
making the same interactions as in the product complex for initation with ATP (Fig. 4A-B, right).
The crystal structure of the initial product complex obtained upon intiation with dpCoA
and CTP (RPo-dpCoApC) shows ordered electron density for the adenosine diphosphate moiety
and two atoms of the pantetheine moiety of the dpCoA residue and for all atoms of the pC residue
(Fig. 4C, left). The dpCoA residue of dpCoApC base pairs to the DNA template strand in the
RNAP "i-1 site," making interactions through its base and phosphates identical to those in the
product complex for initiation with ATP (Fig. 4A,C, right). The first two atoms of the pantetheine
moiety project into a space having sufficient volume to accommodate the pantetheine moiety in
multiple conformational states; terminal atoms of the pantetheine moiety are disordered,
suggesting conformational heterogeneity (flexibility). The pC residue of dpCoApC base pairs to
the DNA template strand in the RNAP active-center “i site,” making the same interactions as in
the product complex for initation with ATP (Fig. 4A,C, right).
Our results establish that NAD+, NADH, and dpCoA function as NCINs for de novo
transcription initiation and establish a mechanism for ab initio, as opposed to post-transcriptioninitiation, capping of cellular RNAs. The results add to an emerging picture that indicates NTPs
are not the only substrates for transcription initiation in vivo (15-17). In addition, the results point
to an urgent need to determine consequences of NAD+-, NADH- and dpCoA-capping for RNA
stability, RNA trafficking, RNA metabolism, and gene expression, both in bacteria and in
eukaryotes.
8
References and Notes:
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I Topisirovic, YV Svitkin, N Sonenberg, AJ Shatkin, Cap and cap-binding proteins in the
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MP Hui, PL Foley, JG Belasco, Messenger RNA degradation in bacterial cells. Annu.
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4.
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H Cahova, ML Winz, K Hofer, G Nubel, A Jaschke, NAD captureSeq indicates NAD as
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DJ Luciano, JG Belasco, NAD in RNA: unconventional headgear. Trends Biochem. Sci.
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7.
A Deana, H Celesnik, JG Belasco, The bacterial enzyme RppH triggers messenger RNA
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MK Thomason, et al., Global transcriptional start site mapping using differential RNA
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P Cramer, Multisubunit RNA polymerases. Curr. Opin. Struc. Biol. 12, 89-97 (2002).
11.
KB Decker, DM Hinton, Transcription regulation at the core: similarities among
bacterial, archaeal, and eukaryotic RNA polymerases. Annu. Rev. Microbiol. 67, 113-139
(2013).
9
12.
Y. Zhang, et al. Structural basis of transcription initiation. Science 338, 1076-1080
(2012).
13.
Y Zhang et al., GE23077 binds to the RNA polymerase 'i' and 'i+1' sites and prevents the
binding of initiating nucleotides. eLife 3, e02450 (2014).
14.
RS Basu, et al., Structural basis of transcription initiation by bacterial RNA polymerase
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15.
SR Goldman, et al., NanoRNAs prime transcription initiation in vivo. Mol. Cell 42, 817825 (2011).
16.
IO Vvedenskaya, et al., Growth phase-dependent control of transcription start site
selection and gene expression by nanoRNAs. Genes Dev. 26, 1498-1507 (2012).
17.
SY Druzhinin, et al., A Conserved Pattern of Primer-Dependent Transcription Initiation
in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq. PLoS Gen. 11,
e1005348 (2015).
18.
W Xu, CR Jones, CA Dunn, MJ Bessman, Gene ytkD of Bacillus subtilis encodes an
atypical nucleoside triphosphatase member of the Nudix hydrolase superfamily. J.
Bacteriol. 186, 8380-8384 (2004).
19.
KD Westover, DA Bushnell, RD Kornberg, Structural basis of transcription: nucleotide
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10
Acknowledgements:
Work was supported by NSF grant CHE-1361462 (JKL), Welch Foundation Grant A-1763
(CDK), and NIH grants GM097260 (CDK), GM041376 (RHE), GM088343 (BEN), and
GM096454 (BEN).
11
Figure Legends
Fig. 1. De novo transcription initiation by ATP and NCINs: initial product formation.
A. Structures of ATP, NAD+, NADH, and dpCoA. Red, identical atoms.
B. Initial RNA products of in vitro transcription reactions with ATP, NAD+, NADH, or dpCoA as
initiating nucleotide and [α32P]-CTP as extending nucleotide (E. coli RNAP; PgadY). Products
were treated with RppH [processes 5'-triphosphate RNA to 5'-monophosphate RNA and 5'-NTP
to 5'-NDP/5'-NMP (7, 18) or NudC [processes 5'-NAD+/NADH-capped RNA to 5'monophosphate RNA (5)] as indicated.
C. LC/MS/MS analysis of initial RNA products of reactions with NAD+ as initiating nucleotide
and CTP as extending nucleotide. Left, NAD+pC (atoms corresponding to CID fragment in red).
Middle, extracted ion chromatogram (signal from detection of parent ion of m/z = 967 and CID
fragment of m/z = 845). Right, MS of CID fragment.
Fig. 2. De novo transcription initiation by ATP and NCINs: full-length product formation.
A. Template comprising PrnaI linked to A-less cassette (promoter elements and start site in gray).
B, C. Full-length RNA products of in vitro transcription reactions with ATP, NAD+, NADH, or
dpCoA as initiating nucleotide and [α32P]-CTP, GTP, and UTP as extending nucleotides (B), or
with [γ32P]-ATP or [α32P]-NAD+ as initiating nucleotide and CTP, GTP, and UTP as extending
nucleotides (C) (E. coli RNAP; PrnaI template shown in panel A). Products were treated with
NudC as indicated. M, 100-nt marker.
Fig. 3. Promoter sequence determines efficiency of NCIN-mediated transcription initiation.
A. Promoters analyzed (promoter elements and start sites in gray).
B. NCIN-capping requires A+1. Initial RNA products of in vitro transcription reactions with ATP,
NAD+, NADH, or dpCoA as initiating nucleotide and [α32P]-CTP as extending nucleotide [E. coli
12
RNAP; wt (+1A), PrnaI (upper) or PgadY (lower); mut (+1G), +1G derivative of PrnaI (upper) or
PgadY (lower)].
C. Promoter sequence determinants in addition to A+1 affect NCIN-capping. Left, initial RNA
products of in vitro transcription reactions with 50 µM ATP or 1 mM NAD+ as initiating
nucleotide and [α32P]-CTP as extending nucleotide (E. coli RNAP; PrnaI, PgadY, PN25, or PT7A1).
Center, dependence of NAD+-capping on [NAD+]/[ATP] ratio (mean±SEM of 4-6
determinations). Right, relative efficiencies of NAD+-capping.
Fig. 4. Structural basis of NCIN-mediated transcription initiation.
A-C. Crystal structures of product complexes for ATP-mediated (A), NAD+-mediated (B), and
dpCoA-mediated (C) transcription initiation (RPo-pppApC, RPo-NAD+pC, and RPo-dpCoApC).
Left, electron density and atomic model for initial RNA product. Green mesh, Fo-Fc omit map
(contoured at 2.5σ in A-B; contoured at 2.2σ in C); red, DNA; pink, RNA product and
diphosphate in "E site" (see 13, 14, 19); violet spheres, Mg2+(I) and Mg2+(II); gray, RNAP bridge
helix. Right, contacts between RPo and initial RNA product. Green and orange, carbon and
phosphorus atoms derived from initiating nucleotide; pink, atoms derived from extending
nucleotide; red, DNA atoms and non-DNA oxygen atoms; blue, nitrogen atoms; gray sticks,
RNAP carbon atoms; violet sphere, Mg2+(I); gray ribbon, RNAP bridge helix.
13
dpCoA
NAD+
NADH
dpCoA
– +
– +
NAD+p*C
– +
+ RppH
+ NudC
– +
NADH
+ RppH
+ NudC
ATP
NAD+
+ RppH
+ NudC
B
ATP
+ RppH
+ NudC
A
dpCoAp*C
NADHp*C
pAp*C
pAp*C
pAp*C
pppAp*C
p*C
p*C
p*C
p*C
pp*C
ppp*C
pp*C
ppp*C
pp*C
ppp*C
pp*C
ppp*C
pAp*C
C
intensity
200
100
0
0
10
time (min)
20
m/z = 845.1
100
relative absorbacne
10.4 min
m/z = 967; CID m/z = 845
300
50
0
200
400
Figure 1
14
600
m/z
800
1000
–10
–35
+1
cTTGAAGtcatgcgccggttaaggcTAAACTgaaaggA
M
NudC
ATP
NAD+
run-off
+112
NADH
dpCoA
NudC
B
A-less
NudC
rnaI
NudC
A
100-nt
C
NudC
M
NudC
[32P]-ATP [32P]-NAD+
100-nt
Figure 2
15
rnaI
–35
–10
+1
aatggCTGATCttatttccagtaaaagtTATATTtaacttA
gadY
–35
–10
+1
tttatTTGCTTtcaggaaaatttttctgTATAATagattcA
N25
–35
–10
+1
agtaTTGACTtaaagtctaacctatagGATACTtacagccA
T7A1
control +1A
promoters
dpCoA
wt (+1A)
wt (+1A)
wt (+1A)
promoters of
NAD+-capped
RNAs
mut (+1G)
NADH
mut (+1G)
NAD+
mut (+1G)
ATP
wt (+1A)
B
–35
–10
+1
agtcTTGAAGtcatgcgccggttaaggcTAAACTgaaaggA
mut (+1G)
A
rnaI
NAD+p*C
pppAp*C
proportion of NAD+ initiation
NAD+p*C/(NAD+p*C + pppAp*C)
C
rnaI
gadY
N25
T7A1
gadY
relative efficiency
(kcat/Km,NAD+)/(kcat/Km,ATP)
1.0
rnaI
gadY
N25
rnaI
0.28 ± 0.03
gadY 0.071 ± 0.01
0.5
N25
T7A1
0.015 ± 0.001
T7A1 0.0047 ± 0.0004
0
1
10
100
[NAD+]/[ATP]
1000
Figure 3
16
Figure 4
17
SUPPLEMENTARY MATERIALS
The mechanism of RNA 5' capping with NAD+, NADH, and CoA
Jeremy G. Bird1,2,*, Yu Zhang2,*, Yuan Tian3, Landon Greene4, Min Liu5, Brian Buckley5,
Jeehiun K. Lee3, Craig D. Kaplan6, Richard H. Ebright2,†, Bryce E. Nickels1,†
File includes:
Materials and Methods
Supplemental References
Figs. S1 to S4
Tables S1 and S2
18
MATERIALS AND METHODS
Proteins
E. coli RNAP core enzyme was prepared from BL21(DE3) cells transformed with
plasmid pVS10 (gift of I. Artsimovitch) as described in (20). σ70 was prepared from BL21(DE3)
cells transformed with plasmid pσ70-His (gift of J. Roberts) as described in (21, 22). RppH was
purchased from NEB. NudC was purified from BL21(DE3) cells from pET NudC-His, which was
prepared as described in (5). Briefly, nudC was amplified by PCR from E. coli strain MG1655
using oligonucleotides JB221 and JB222. The PCR product was digested with XbaI and NotI and
inserted into pET28c digested with XbaI and NotI. T. thermophilus RNAP holoenzyme was
prepared as in (12). Saccharomyces cerevisiae RNAP II was prepared as in (23).
Oligonucleotides
Sequences of oligonucleotides used in this work are listed in Table S2.
In vitro transcription assays with E. coli RNAP: template preparation
Linear transcription templates were synthesized by PCR using Phusion HF Polymerase
master mix (Thermo) and oligonucleotides listed in Table S2. Wt gadY (+1A) template used in
Figs. 1B-C, 3B-C, S1: JB224, JB230, JB231; rnaI A-less cassette template used in Fig. 2: JB250,
JB248 and JB251; rnaI wt (+1A) template used in Fig 3B,C: JB250, JB248 and JB269; rnaI mut
(+1G) template used in Fig. 3B: JB258, JB248 and JB269; gadY mut (+1G) template used in Fig.
3B: JB244, JB230, JB231; N25 template used in Fig. 3B: JB270, JB233 and JB234; T7A1
template used in Fig. 3B: JB235, JB228 and JB229.
In vitro transcription assays with E. coli RNAP: abortive initiation assays
10 nM of linear template was mixed with 50 nM core RNAP and 250 nM σ70 in 60 µl
transcription buffer (10 mM Tris HCl pH 8.0, 40 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM
DTT, 0.1 mg/ml BSA, 2% glycerol) and incubated at 37°C for 15 minutes to form open
complexes. The indicated initiating nucleotide [0.2 mM ATP (Roche), 1 mM NAD+ (Roche), 1
mM NADH (Roche) or 1 mM dpCoA (Sigma-Aldrich)] was added along with 6 µCi [α32P]-CTP
(Perkin Elmer; 3000 Ci/mmol) and reactions were incubated at 37°C for 10 minutes to allow for
product formation. For the NAD+ /ATP competition assays shown in Fig. 2C, 1 mM NAD+ was
present in each reaction along with a varying concentration of ATP. For experiments shown in
Figs. 2B and 2C, reactions were stopped by addition of an equal volume of gel loading buffer
[90% formamide, 100 mM Tris-HCl pH 8.0, 18 mM EDTA, 0.025% xylene cyanol, 0.025%
bromophenol blue]. For the reactions shown in Fig. 1B the samples were passed through a
Nanosep Centrifugal Device (10 kDa molecular weight cutoff; Pall Corporation), the flow
through was collected, mixed with 1 Unit RppH (NEB), 400 nM NudC or NudC storage buffer
(50 mM Tris HCl pH 7.9, 0.5M NaCl, 50 mM KCl, 1 mM MgCl2, 7mM TCEP, 50% glycerol),
incubated at 37°C for 30 minutes, and mixed with an equal volume of gel loading buffer. Samples
were run on 15% TBE-Urea polyacrylamide gels (UreaGel system, National Diagnostics).
Autoradiography of gels was performed using storage phosphor screens and a Typhoon 9400
variable mode imager (GE Life Science) and quantified using ImageQuant software. We note that
the presence of contaminating AMP in the dpCoA stock (Fig. S4) leads to the generation of pApC
products in reactions performed with dpCoA.
In vitro transcription assays with E. coli RNAP: run-off transcription assays
For reactions shown in Figure S2B, open complexes formed as described above were
mixed with the indicated initiating nucleotide [0.2 mM ATP, 1 mM NAD+, 1 mM NADH or 1
mM dpCoA] along with 6 µCi [α32P]-CTP (Perkin Elmer; 3000 Ci/mmol), 200 µM CTP, 200 µM
19
UTP, and 200 µM GTP, and reactions were incubated at 37°C for 10 minutes to allow for product
formation. For the reactions shown in Figure S2C, open complexes formed as described above
were mixed with 10 µCi [γ32P]-ATP (Perkin Elmer; 6000 Ci/mmol), or 20 µCi [α32P]-NAD+
(Perkin Elmer; 800 Ci/mmol) along with 200 µM CTP, 200 µM UTP and 200 µM GTP.
Reactions were incubated at 37°C for 10 minutes to allow for product formation, and stopped by
addition of 100 µl stop solution (0.6 M Tris HCl pH 8.0, 18 mM EDTA, 0.1 mg/ml glycogen).
Samples were extracted with acid phenol:chloroform and RNA transcripts were recovered by
ethanol precipitation and resuspended in 20 µl of transcription buffer and divided into two 10 µl
aliquots. 400 nM NudC was added to one aliquot while NudC storage buffer was added to the
other for mock treatment. Reactions were incubated at 37°C for 30 minutes and stopped by
addition of an equal volume of gel loading buffer and analyzed by gel electrophoresis on 15%
TBE-Urea polyacrylamide gels as described above. Reactions were loaded alongside an Ambion
Decade Marker System (Life Techniologies) to enable an estimation of the approximate size of
the full-length transcripts generated.
In vitro transcription assays with E. coli RNAP: liquid-chromatography/tandem-mass
spectrometry (LC/MS/MS) detection of NAD+pC
Open complexes formed as described above were mixed with 2 mM NAD+ and 10 mM
CTP, incubated at 37°C for 1 hour, passed through a Nanosep Centrifugal Device, and the flow
through was analyzed by LC/MS/MS. (Control samples in which NAD+, CTP, or RNAP were not
present in the reactions were also prepared.) LC/MS/MS analyses were performed in negative
ion mode with a Finnigan LTQ mass spectrometer equipped with an electrospray ionization (ESI)
interface coupled with a Finnigan Surveyor HPLC system. The flow rate from the Finnigan
Surveyor pump was 0.3 ml/min. The autosampler temperature was 5°C. The liquid
chromatography method used a YMC ODS-A 5 µm particle size 120Å pore size column, 3.0 mm
x 100 mm. The samples were separated using a gradient mobile phase consisting of 5 mM
ammonium formate buffered to pH 7.9 in water (A) and methanol (B). The gradient condition
was: 0–5 min, 100% A; 5-15 min, 100-30% A; 15-23 min, 30-0% A; 23-28 min, 0% A; 28-40
min, 100% A. Column temperature was 25°C. The temperature of the heated capillary was
200°C. Fragmentation was activated by collision induced dissociation (CID) with a collision
energy of 20-25%. The search for NAD+pC was conducted by isolating its anion [(M–2H), m/z
967] at isolation width 3 using full scan mode, and analyzing its fragmentation spectrum
(collision energy 20%) from m/z = 210 to m/z = 1000. The characteristic fragment m/z = 845
(loss of nicotinamide) was used to identify NAD+pC. The instrument control, data acquisition,
and data analysis were performed by Xcalibur software. Ammonium formate (99%), water
(HPLC grade) and methanol (HPLC grade) used in these LC/MS/MS experiments were
purchased from Sigma-Aldrich.
In vitro transcription assays with S. cerevisiae RNAP II
Bubble templates for initiation were utilized following the approach in (24) by mixing 2.5
nmol oligo CKO1639 and 2.5 nmol CKO1621 in 100 µl of annealing buffer (40 mM Tris pH 8.0,
50 mM NaCl, 1 mM EDTA), incubating at 95°C for 3 minutes, and slowing cooling to 23°C
(1°C/minute) to allow annealing. The oligo mixture was run on a 2.5% agarose gel (Apex,
Genesee) equilibrated and run in 1x TBE [90 mM Tris base (BioRad), 90 mM boric acid
(Calbiochem), 2 mM EDTA (JT Baker)], the gel was stained with Ethidium Bromide and the
band corresponding to the bubble template was excised from the gel, crushed, and incubated with
500 µl elution buffer (500 mM NH4OAc, 10 mM MgOAc, 1 mM EDTA, 0.1% SDS). Gel debris
was removed using a Spin-X column (Costar) and nucleic acids were isolated by ethanol
precipitation and re-suspended in nuclease-free water (Ambion) to a concentration of 0.5 µg/µl.
500 ng of the template was mixed with M280 magnetic streptavidin beads (Invitrogen) in 1x TE
20
(from Ambion components) supplemented with 1 M NaCl (EMD/Calbiochem) and incubated at
25°C for 30 minutes to allow binding. Templates bound to the beads were washed three times
using magnetic capture to separate beads in 1x TE supplemented with 1 M NaCl, once with 1x
TE, and then equilibrated into 1x transcription buffer [TB: 20 mM Tris pH 8.0 (Ambion), 40 mM
KCl (Ambion), 10 mM MgCl2 (Ambion), 10% glycerol (Macron/Avantor), BSA 0.25 mg/ml
(Ambion), 1 mM DTT (Gold Biotechnology)].
2.5 ug purified Saccharomyces cerevisiae RNAP II was added to each template in 50 µl
1x TB, and incubated with rotation at 25°C for 20 minutes. Complexes were washed following
magnetic capture twice with 200 µl 1x TB, and resuspended in 22 µl 2x TB containing 0.4 U/µl
RNase Inhibitor (NEB). 7.5 µl of this mixture was aliquoted for reactions (4 reactions per bubble
template).
5 µl of "initiator"/ [α32P]-UTP mixes were added to give final concentrations of: 1 mM
ATP (GE Lifesciences), 2.5 µM [α32P]-UTP (from 800 Ci/mmol [12.5 µM], Perkin Elmer); 1
mM NAD+, 2.5 µM [α32P]-UTP (from 800 Ci/mmol [12.5 µM], Perkin Elmer); or 1mM NADH,
2.5 µM [α32P]-UTP (from 800 Ci/mmol [12.5 µM], Perkin Elmer). Reactions were incubated at
25°C for 2 hours then passed through a Nanosep Centrifugal Device (10 kDa molecular weight
cutoff). The flow through was collected, mixed with 0.5 µl (2.5 U) RppH (NEB), 0.5 µl NudC
(400 nM final) or 0.5 µl NudC storage buffer, incubated at 37°C for 30 minutes, and mixed with
an equal volume of urea gel loading buffer [10M Urea (Calbiochem), 0.5 M EDTA pH 8.0 (JT
Baker), 1× TBE]. Samples were run on 25% 19:1 acrylamide:bisacrylamide gel [from 40% 19:1
solution (BioRad), 7 M Urea (Calbiochem), 1x TBE (90 mM Tris base; BioRad), 90 mM boric
acid (Calbiochem), 2 mM EDTA (JT Baker)]. Autoradiography of gels was performed using
storage phosphor screens and a Pharos (Bio-Rad) imager. Radiolabeled products were visualized
using ImageLab (Bio-Rad) software.
Structure determination: RPo-pppApC
Crystals of T. thermophilus RPo were prepared using a derivative of the nucleic-acid
scaffold used for analysis of RPo in (12) having A in place of C at position -1 of the DNA
template strand, and were grown and handled essentially as in (12). Crystallization drops
contained 1 µl RPo in 20 mM Tris-HCl, pH 7.7, 100 mM NaCl, and 1% glycerol, and 1 µl
reservoir buffer (RB; 100 mM Tris-HCl, pH 8.4, 200 mM KCl, 50 mM MgCl2, and 9.5%
PEG4000), and were equilibrated against 400 ml RB in a vapor-diffusion hanging-drop tray. Rodlike crystals appeared in 1 day, and were used to micro-seed hanging drops using the same
conditions. ATP and CTP (Sigma-Aldrich) were soaked into RPo crystals by addition of 0.2 µl 40
mM ATP and 40 mM CTP in 60% (v/v) RB to the crystallization drop, and incubated for 2 min at
22°C. Crystals were transferred into reservoir solutions containing 2 mM ATP, 2 mM CTP, and
17.5% (v/v) (2R, 3R)-(-)-2,3-butanediol (Sigma-Aldrich) and were flash-cooled with liquid
nitrogen.
Diffraction data were collected at BNL beamline X29A, processed and scaled using
HKL2000 (25). Structure factors were converted using the French-Wilson algorithm in Phenix
(26) and were subjected to anisotropy correction using the UCLA MBI Diffraction Anisotropy
server [(27); http://services.mbi.ucla.edu/anisoscale/]. The structure was solved by molecular
replacement with Molrep (28) using one RNAP molecule from the structure of T. thermophilus
RPo [PDB 4G7H; (12)] as the search model. Early-stage refinement included rigid-body
refinement of each RNAP molecule, followed by rigid-body refinement of each subunit of RNAP
molecules. Cycles of iterative model building with Coot (29) and refinement with Phenix (30)
were performed. Atomic models of the DNA nontemplate strand, the DNA template strand, and
RNA were built into mFo-DFc omit maps, and subsequent cycles of refinement and model
building were performed. The final crystallographic model of RPo-pppApC, refined to Rwork and
Rfree of 0.211 and 0.258, has been deposited in the PDB with accession code 5D4C.
21
Structure determination: RPo-NAD+pC
NAD+ (Sigma-Aldrich) and CTP were soaked into RPo crystals (prepared as described
above) by addition of 0.2 µl 30 mM NAD and 40 mM CTP in 50% (v/v) RB to the crystallization
drop, and incubated for 120 min at 22°C. Crystals were transferred into reservoir solutions
containing 1.5 mM NAD, 2 mM CTP, and 17.5% (v/v) (2R, 3R)-(-)-2,3-butanediol and were
flash-cooled with liquid nitrogen.
Diffraction data were collected at APS beamline 19-ID-D, processed and scaled using
HKL2000 (25), and subjected to anisotropic correction using the UCLA MBI Diffraction
Anisotropy server [(27); http://services.mbi.ucla.edu/anisoscale/]. The structure was solved and
refined using procedures analogous to those described above for RPo-pppApC. The final
crystallographic model of RPo-NAD+pC, refined to Rwork and Rfree of 0.201 and 0.254,
respectively, has been deposited in the PDB with accession code 5D4D.
Structure determination: RPo-dpCoApC
dpCoA (Sigma-Aldrich) and CTP were soaked into RPo crystals (prepared as described
above) by addition of 0.2 µl 60 mM 3'-dephosphate-CoA and 40 mM CTP in 50% (v/v) RB to the
crystallization drop, and incubated for 120 min at 22°C. Crystals were transferred into reservoir
solutions containing 3 mM 3'-dephosphate-CoA, 2 mM CTP, and 17.5% (v/v) (2R, 3R)-(-)-2,3butanediol and were flash-cooled with liquid nitrogen.
Diffraction data were collected at APS beamline 19-ID-D, processed and scaled using
HKL2000 (25), and subjected to anisotropic correction using the UCLA MBI Diffraction
Anisotropy server [(27); http://services.mbi.ucla.edu/anisoscale/]. The structure was solved and
refined using procedures analogous to those described above for RPo-pppApC. The final
crystallographic model of dpCoApC, refined to Rwork and Rfree of 0.210 and 0.247, respectively,
has been deposited in the PDB with accession code 5D4E.
Analysis of dpCoA by liquid chromatography (LC)-UV
AMP (Sigma-Aldrich) and dpCoA were diluted to 1 mM in water (HPLC grade) before
injection. LC-UV analyses were performed using a Waters 2960 HPLC system coupled with a
Waters 2996 photodiode array detector. The flow rate from the Finnigan Surveyor pump was 0.3
ml/min. The autosampler temperature was 5°C. The LC method used an YMC ODS-A 5 µm
particle size 120Å pore size column, 3.0 mm x 100 mm. The samples were separated using a
gradient mobile phase consisting of 25 mM triethylammonium formate buffered to pH 3.0 in
water (A) and methanol (B). The gradient condition was: 0-5 min, 100% A; 5-15 min, 100-30%
A; 15-23 min, 30-0% A; 23-28 min, 0% A; 28-40 min, 100% A. Column temperature was 25 °C.
Detector wave length was 260 nm. The instrument control, data acquisition, and data analysis
were performed by MassLynx software. Triethylammonium acetate buffer (2.0 M) and formic
acid (>95%) were purchased from Sigma-Aldrich.
22
SUPPLEMENTAL REFERENCES
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
I. Artsimovitch, V. Svetlov, K. S. Murakami, R. Landick, Co-overexpression of
Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant
enzymes lacking lineage-specific sequence insertions. J. Biol. Chem. 278, 12344-12355
(2003).
M. T. Marr, J. W. Roberts, Promoter recognition as measured by binding of polymerase
to nontemplate strand oligonucleotide. Science 276, 1258-1260 (1997).
S. A. Perdue, J. W. Roberts, A backtrack-inducing sequence is an essential component of
Escherichia coli σ70-dependent promoter-proximal pausing. Mol. Microbiol. 78, 636-650
(2010).
C. D. Kaplan, K. M. Larsson, R. D. Kornberg, The RNA polymerase II trigger loop
functions in substrate selection and is directly targeted by alpha-amanitin. Mol. Cell 30,
547-556 (2008).
P. Cabart, H. Jin, L. Li, C. D. Kaplan, Activation and reactivation of the RNA
polymerase II trigger loop for intrinsic RNA cleavage and catalysis. Transcription 5,
e28869 (2014).
Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in oscillation
mode. Method Enzymol. 276, 307-326 (1997).
S. French, K. Wilson, On the treatment of negative intensity observations. Acta
Crystallographica Section A 34, 517-525 (1978).
M. Strong, M. R. Sawaya, S. Wang, M. Phillips, D. Cascio, D. Eisenberg, Toward the
structural genomics of complexes: crystal structure of a PE/PPE protein complex from
Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 103, 8060-8065 (2006).
A. Vagin, A. Teplyakov, MOLREP: an Automated Program for Molecular Replacement.
J Appl. Crystallogr. 30, 1022-1025 (1997).
P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot.
Acta Crystallographica. Section D, Biological Crystallography 66, 486-501 (2010).
P. D. Adams et al., PHENIX: a comprehensive Python-based system for macromolecular
structure solution. Acta Crystallographica Section D Biological Crystallography 66, 213221 (2010).
23
B
intensity
A
10.4 min
m/z = 967; CID m/z = 845
300
200
RNAP, DNA, NAD+, CTP
100
intensity
0
300
200
RNAP, DNA, NAD+
100
intensity
0
300
200
RNAP, DNA, CTP
100
intensity
0
300
200
DNA, NAD+, CTP
100
0
0
10
20
time (min)
C
40
m/z = 845.1
100
relative absorbacne
30
50
0
200
400
600
800
1000
m/z
Fig. S1. LC/MS/MS analysis of initial RNA products of in vitro transcription reactions with
NAD+ as initiating nucleotide and CTP as an extending nucleotide.
A. Structure of NAD+pC (red, atoms corresponding to CID-generated fragment ion).
B. Extracted ion chromatogram (signal derived from detection of parent ion of m/z = 967 and
CID fragment of m/z = 845 corresponding to NAD+pC minus nicotinamide). Reactions contained
the indicated components.
C. Mass spectrum of CID fragment.
24
NAD+
enrichment
–35
–10
+1
TTGAAGtcatgcgccggttaaggcTAAACTgaaaggA rnaI
588
–35
–10
+1
TTGAACttttccggggcatataacTATACTccccgcA copA
549
–35
–10
+1
CTGATCttatttccagtaaaagtTATATTtaacttA gadY
104
–35
–10
+1
TTGAGCttctaccagcaaataccTATAGTggcggcA gcvB
52
–35
–10
+1
TTGGCAggatggtgagattgagcGACAATcgagttA chiX
52
–35
–10
+1
TTGGCAactaaaggttaaaaccgtTATAACacagtcA mcaS
24
plasmid-encoded
chromosome-encoded
Fig. S2. Promoters for full-length RNAs identified as being enriched for NAD+-capping in
vivo.
NAD+ enrichment levels as determined in E. coli strain JM109 (5). Promoter elements and
transcription start sites are in gray.
25
NudC
RppH
NADH
NudC
RppH
NAD+
NudC
RppH
ATP
NAD+p*U
NADHp*U
pAp*U
pAp*U
ppp*U
ppp*U
pAp*U
pppAp*U
ppp*U
Fig S3. NCIN-mediated de novo transcrption initiation by eukaryotic RNAP II.
Initial RNA products of in vitro transcription reactions with ATP, NAD+, or NADH as initiating
nucleotide and [α32P]-UTP as extending nucleotide. Reactions were performed with yeast RNAP
II and an artificial-bubble transcription initiation template. Products were treated with RppH or
NudC as indicated.
26
AMP
dpCoA
7.05
13.42
3
AU
2
1
7.11
0
0
5
10
15
20
time (min)
25
30
35
40
Fig. S4. AMP content of dpCoA stock.
HPLC chromatogram of dpCoA stock (Sigma-Aldrich, lot SLBJ2886V; 50 nmol). Green: HPLC
chromatogram of AMP (20 nmol). Comparison of chromatograms indicates that the dpCoA stock
contains ~2% AMP. The observation that the dpCoA stock contains ~2% AMP in the dpCoA
stock accounts for the formation of pApC in reactions performed with dpCoA (Fig. 1B).
27
Table S1. Structure data collection and refinement statistics
dataset
RPo-pppApC
RPo-NADpC
beamline
BNL-X29A
APS-19-ID
RPo-3’-dephosphateCoApC
APS-19-ID
space group
P2 (1)
P2 (1)
P2 (1)
resolution range
completeness
50.00-3.30 Å (3.36-3.3050.00-3.00 Å (3.05-3.00 40.00-3.10 Å (3.15-3.10
Å)
Å)
Å)
a=186.0, b=103.6,
a=185.0, b=103.5,
a=185.8, b=103.9,
c=297.4
c=296.3
c=297.1
α=90.0, β=98.3, γ=90.0 α=90.0, β=98.3, γ=90.0 α=90.0, β=98.5, γ=90.0
0.961 (0.875)
0.979 (0.934)
0.989 (0.960)
multiplicity
5.4 (5.0)
4.0 (3.7)
3.8 (3.5)
mean I/σ
Rmerge
8.7 (1.7)
10.6 (1.5)
10.4 (1.4)
0.185 (0.954)
0.119 (0.920)
0.120 (0.840)
Rwork
0.211
0.201
0.210
Rfree
0.258
0.254
0.247
bond-length rmsd
0.004 Å
0.008 Å
0.004 Å
bond-angle rmsd
0.791°
0.952°
0.698°
PDB code
5D4C
5D4D
5D4E
cell parameters
28
Table S2. Oligonucleotides
Name
JB 221
Description
NudC-XbaI forward primer
JB 222
NudC-NotI reverse primer
JB 224
gadY wild-type promoter
template (-65 to +35)
JB 228
T7A1 promoter forward primer
JB 229
JB 230
JB 231
JB 233
T7A1 promoter reverse primer
gadY forward primer
gadY reverse primer
N25 forward primer
JB 234
JB 235
N25 reverse primer
T7A1 (+2C,+3T) promoter
template (-65 to +35)
JB 244
gadY (+1G) promoter template
DNA
JB 248
JB 250
RNAI forward primer
RNAI wild-type promoter
template (-65 to +112, A-less)
JB 251
RNAI reverse primer (+112)
JB 258
RNAI (+1G) promoter template
(-65 to +112, A-less)
JB 269
JB 270
RNAI reverse primer (+35)
N25 (+2C, +3T) promoter
template (-65 to +35)
Sequence (5' to 3')
CAATTCCCCTCTAGAAATAATTTTGTTTA
ACTTTAAGAAGGAGATATAATGGATCGT
ATAATTGAAAAATTAGATCACGGC
GTGCTCGAGTGCGGCCGCGCTGCCGCGC
GGCACCAGCTCATACTCTGCCCGACACA
TCGCCACCGT
AGCGTATAGCTTATGTTTATAAAAAAAT
GGCTGATCTTATTTCCAGTAAAAGTTATA
TTTAACTTACTGAGAGCACAAAGTTTCC
CGTGCCAACAGGGAG
GATTAATTTAAAATTTATCAAAAAGAGT
ATTGAC
TCGTTGGGATGGCTA
AGCGTATAGCTTATG
CTCCCTGTTGGCACGGGAAAC
ATCCGTCGAGGGAAATCATAAAAAATTT
ATTTGC
AACCAGCCATATTTAAACTCCTC
GATTAATTTAAAATTTATCAAAAAGAGT
ATTGACTTAAAGTCTAACCTATAGGATA
CTTACAGCCaCTGAGAGGGACACGGCGA
ATAGCCATCCCAACGA
AGCGTATAGCTTATGTTTATAAAAAAAT
GGCTGATCTTATTTCCAGTAAAAGTTATA
TTTAACTTGCTGAGAGCACAAAGTTTCC
CGTGCCAACAGGGAG
CCACTGGCAGCAGCCACTGG
CCACTGGTAATTGATTTAGAGGAGTTAG
TCTTGAAGTCATGCGCCGGTTAAGGCTA
AACTGAAAGGACTTGTTTTGGTGTCTGC
GCTCCTCCTTGCCTGTTTCCTCGGTTCTTT
GTGTTGGTTGCTCTGTGTTCCTTCGTTTTT
CCGCCCTGCTTGGCGGTTTTTTCGTTTTC
TGTGC
GCACAGAAAACGAAAAAACCGCCAAGC
AGG
CCACTGGTAATTGATTTAGAGGAGTTAG
TCTTGAAGTCATGCGCCGGTTAAGGCTA
AACTGAAAGGGCTTGTTTTGGTGTCTGC
GCTCCTCCTTGCCTGTTTCCTCGGTTCTTT
GTGTTGGTTGCTCTGTGTTCCTTCGTTTTT
CCGCCCTGCTTGGCGGTTTTTTCGTTTTC
TGTGC
AACAGGCAAGGAGGAGCGCAG
ATCCGTCGAGGGAAATCATAAAAAATTT
ATTTGCTTTCAGGAAAATTTTTCTGTATA
ATAGATTCACTAATTTGAGAGAGGAGTT
29
CK01639
Bubble template, template strand
CK01621
Bubble template, non-template
strand (carries 5' biotin)
30
TAAATATGGCTGGTT
TGAAGTCTTGTGTGGTCCTGAGAAAGTG
TTGAGATCCATGACAGAAAGATTAATAA
TTGTATGACTATTTATACGCGTCCTGT
Biotin/ACAGGACGCGTATAAATAGTCATA
CAATTATTAATCTTTCACGATCTTTCCTC
AACACTTTCTCAGGACCACACAAGACTT
CA